RNA-mediated gene assembly from DNA oligonucleotides

Patent metadata

Field	Value
Publication number	US-9873900-B2
Application number	US-201313763009-A
Country	US
Kind code	B2
Filing date	Feb 8, 2013
Priority date	Feb 10, 2012
Publication date	Jan 23, 2018
Grant date	Jan 23, 2018

Abstract

The present invention is directed to methods and materials for RNA-mediated gene assembly from oligonucleotide sequences. In some embodiments, the oligonucleotides used for gene assembly are provided in an array format. An RNA polymerase promoter is appended to surface-bound oligonucleotides and a plurality of RNA copies of each oligonucleotide are then produced with an RNA polymerase. These RNA molecules self-assemble into a desired full-length RNA transcript by hybridization and ligation. The resulting RNA transcript may then be converted into double strand DNA useful in a variety of applications including protein expression.

First claim

What is claimed is:

1. An oligonucleotide array for RNA-mediated assembly of a target RNA molecule, comprising:
(a) a plurality of first surface-bound oligonucleotides each having a segment sequence corresponding to a portion of a target RNA and a complementary RNAP promoter sequence operably-linked to said segment sequence's 3′ termini, wherein two or more of the segment sequences correspond to different portions of the target RNA; and
(b) a plurality of second surface-bound oligonucleotides each having a splint sequence corresponding to a portion of the target RNA that complements and partially overlaps at least two of the two or more segment sequences of the first surface-bound oligonucleotides corresponding to different portions of the target RNA, said second surface-bound oligonucleotides including an RNAP promoter sequence operably-linked to their splint sequence's 3′ termini,
wherein said first and second surface-bound oligonucleotides are linked at their 3′ termini to a surface of the oligonucleotide array.

2. The oligonucleotide array according to claim 1 , wherein the 5′ end of each segment sequence and each splint sequence corresponds to a GG dinucleotide in the target RNA molecule.

3. The oligonucleotide array according to claim 1 , wherein the target RNA molecule is a full-length RNA transcript of a gene.

4. The oligonucleotide array according to claim 1 , wherein the oligonucleotide array's surface comprises a silanized glass or amorphous carbon deposited on a gold film.

5. The oligonucleotide array according to claim 1 , wherein said surface-bound oligonucleotides include a spacer, a T7 RNAP promoter sequence, a CC dinucleotide and either the segment sequence or the splint sequence.

6. The oligonucleotide array according to claim 1 , wherein oligonucleotide array further comprises a third oligonucleotide encoding an RNAP promoter sequence to the complementary RNAP promoter sequence of the first and second surface-bound oligonucleotides to yield double-stranded RNAP promoters.

Assignees

• Wisconsin Alumni Res Found

Inventors

• Wu Cheng-Hsien
• Smith Lloyd

Classifications

• C40B40/08

  Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries · CPC title

• C12P19/34 Primary

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title